Unveiling the mechanism of action of acylated temporin L analogues against multidrug-resistant Candida albicans

Abstract The increasing resistance of fungi to conventional antifungal drugs has prompted worldwide the search for new compounds. In this work, we investigated the antifungal properties of acylated Temporin L derivatives, Pent-1B and Dec-1B, against Candida albicans, including the multidrug-resistant strains. Acylated peptides resulted to be active both on reference and clinical strains with MIC values ranging from 6.5 to 26 µM, and they did not show cytotoxicity on human keratinocytes. In addition, we also observed a synergistic or additive effect with voriconazole for peptides Dec-1B and Pent-1B through the checkerboard assay on voriconazole-resistant Candida strains. Moreover, fluorescence-based assays, NMR spectroscopy, and confocal microscopy elucidated a potential membrane-active mechanism, consisting of an initial electrostatic interaction of acylated peptides with fungal membrane, followed by aggregation and insertion into the lipid bilayer and causing membrane perturbation probably through a carpeting effect.


Introduction
Fungal diseases affect billions of individuals and represent a worldwide threat to human health. The significant increase of fungal infections is likely due to the increased use of immunosuppressive therapies in cancer or transplantation, as well as medical devices and the indiscriminate consumption of antibiotics. Antifungal treatments are essential for reducing comorbidities and mortalities, but resistance-related issues have prompted the search for more effective alternatives with reduced toxicity, improved pharmacodynamics and pharmacokinetics, and increased specificity 1 . Candidiasis is the most notable infection caused by fungi of the Candida genus and represents a major concern for public health 2 . Species of Candida are the leading pathogens causing around 400,000 systemic fungal diseases worldwide 3 and are considered the fourth most common cause of nosocomial blood infections, even overcoming Gram-negative pathogens 4,5 . Although more than one hundred Candida species exist, only a few are implicated in clinical infections. Of these, C. albicans is certainly the most isolated species from clinical specimens and it is generally found in 90-100% of mucosal isolates and 50-70% of blood infection isolates 6 . Candida spp infections exhibit different clinical manifestations, from superficial mucocutaneous candidiasis affecting mucosal, nail and skin surfaces, to systemic infections affecting organs 7 . These infections are commonly seen in immunocompromised patients or in those conditions in which yeast overgrowth is favored 7 . Candida species have developed mechanisms of resistance towards antifungal drugs commonly used for the treatment of systemic or topical infections 8,9 . Among them, azoles (e.g. fluconazole, voriconazole), polyenes (e.g. amphotericin B) and echinocandins (e.g. caspofungin) are the most efficacious as they specifically interfere with the ergosterol or cell wall biosynthesis, which are not present in mammalian cells 8 . For example, activities of the azoles are directed against lanosterol 14-a-demethylase (Erg11p), which is encoded by the ERG11 gene, involved in the ergosterol biosynthesis. Among resistant clinical isolates of C. albicans, ERG11 over-expression is frequently observed 10 . The numerous toxic effects, including liver and kidney dysfunctions, local phlebitis, haemolysis and fever 11,12 caused by these antifungal drugs prompted the focus of recent research on new agents with improved safety profile and broadspectrum antimicrobial activity for use alone and/or in synergy with antifungals.
Emerging drug candidates attracting the attention of researchers are antimicrobial peptides (AMPs), host defence peptides that play an essential role in the innate immune response 12 . AMPs, mostly isolated from living organisms, are typically featured by a net positive charge at physiological pH due to the presence of basic residues (e.g. Arg and Lys) while the hydrophobic residues determine their amphipathic conformation. Both the cationic nature and the secondary structure facilitate the interaction with the negatively charged membrane of microorganisms 12,13 , leading to insertion, destabilisation and disruption of the cell membrane, although they can also act on intracellular targets 14,15 . Since the cell membrane is the primary target of AMPs, microorganisms are less likely to develop a resistance mechanism, making AMPs promising drug candidates 16 .
Temporins are AMPs isolated from the skin of the frog Rana temporaria, and exhibit strong antimicrobial, anti-Candida and antiviral activities [17][18][19] . Temporin L (TL) is the most active isoform on both Gram-negative and Gram-positive bacteria and yeasts, but it has high haemolytic activity 18,20 . Previous structure-activity relationship studies (SARs) carried out by our research group led to the discovery of synthetic peptides derived from Temporin L possessing a strong ability to kill both Gram-positive and Gramnegative bacteria, and yeasts as Candida spp 21 . Interestingly, some of them have also demonstrated scarce or absent haemolytic activity at their antimicrobial concentrations 21,22 . Our group has deeply worked on a promising molecule derived from Temporin L, [Pro 3 DLeu 9 DLys 10 ] TL (namely 1B, sequence: Phe-Val-Pro-Trp-Phe-Ser-Lys-Phe-DLeu-DLys-Arg-Ile-Leu-CONH 2 ), that has proved to be an efficient antimicrobial agent, also devoid of cytolytic effects in vitro 23 . Interestingly, peptide 1B has demonstrated the ability to inhibit the growth of methicillin-resistant Staphylococcus pseudintermedius (MRSP), and Malassezia pachydermatis and the prolonged application of this peptide did not cause M. pachydermatis drug-resistance and increased the susceptibility of MRSP to oxacillin 24 . In addition, 1B has shown anti-inflammatory activity in vivo in response to zymosan-induce peritonitis 25 . This result is worthy of note since it supports a possible application of 1B as an antiinflammatory and antimicrobial agent. These results have also been described in a patent which has granted on 7 May 2021 (Number: 102019000009489).
Approaches to reduce or eliminate the toxicity of conventional antifungal drugs and enhance their activity may include the use of drug delivery systems or novel strategies that likely improve the interaction with the target membranes [26][27][28] .
To improve the antimicrobial profile of peptide 1B, we have previously applied a lipidation strategy 29 consisting of the addition of fatty acids in Nand C-terminus of the peptide, and discovered the analogue Pent-1B, bearing a pentyl chain in para position of Phe 1 (peptide C in Bellavita et al. 29 ). This compound has shown a good activity against carbapenem resistant Klebsiella pneumoniae and a low cytotoxicity against both human erythrocytes and keratinocytes 29,30 .
Through detailed biophysical studies focussed on the mode of action of Pent-1B using bacterial biomembrane models, we demonstrated that Pent-1B has a low ability to aggregate in aqueous solution, while it has a significant ability to oligomerize in membrane mimetic environments. This causes an alteration of the membrane fluidity and, by creating pores in both Gram-positive and Gram-negative membranes, eventually leads to cell death 29 .
Based on the results until here obtained, herein, we were prompted to investigate the antifungal properties of Pent-1B against reference and clinical strains of C. albicans, and with the further aim to increase the affinity and interaction with fungal membrane, we designed and synthesised the peptide Dec-1B bearing a longer lipid tail of 10-carbon atoms. The antifungal properties of the peptide Dec-1B were investigated against C. albicans, while its cytotoxicity was assessed on human keratinocytes. The effect of Pent-1B and Dec-1B in combination with antifungal drug voriconazole was evaluated on voriconazole-resistant C. albicans strains. Moreover, the mechanism of action on fungal membrane models of peptides Pent-1B and Dec-1B was explored by fluorescence assays, including Thioflavin T and leakage assays, tryptophan quenching experiments, nuclear magnetic resonance (NMR) spectroscopy and confocal microscopy.

Candida strains and culture conditions
The yeast strains evaluated in this study included C. albicans ATCC 90028 (reference strain) and C. albicans ATCC 10231 (azole-resistant strain) obtained from the American Type Culture Collection (Rockville, MD), and six azole-resistant clinical isolates (Ca1-Ca6) belonging to an anonymous collection of fungal strains established at the Department of Molecular Medicine and Medical Biotechnology, University of Naples Federico II (Table 1). Identification was performed by subcultures on chromogenic agar (Chromid agar) (Becton Dickinson) and confirmed by MS MALDI-TOF (Bruker). Susceptibility to amphotericin B, anidulafungin, micafungin, caspofungin, posaconazole, voriconazole, itraconazole, and fluconazole was assessed using the Sensititre Yeast One colorimetric microdilution method (Thermofisher). The results were interpreted according to the MIC breakpoints reported by Clinical and Laboratory Standards Institute (CLSI) 31 with the exception of the MICs of itraconazole, posaconazole and amphotericin B, which were interpreted according to European Committee on Antimicrobial Susceptibility Testing (Eucast) 32 . All strains were stored as 15% (v/v) glycerol stocks at À80 C. Prior to each experiment, cells were subcultured from the stocks onto Sabouraud dextrose agar (SDA) (Becton Dickinson) at 37 C for 48 h.

Acylated peptide synthesis
The acyl-peptides Pent-1B and Dec-1B were synthesised by using ultrasounds-assisted solid-phase peptide synthesis (US-SPPS) 33 and by employing the Rink amide resin as solid support 29 . In details, peptide assembly was obtained by performing repeated cycles of (i) Fmoc deprotections, by treatments with 20% of piperidine in DMF solution [0.5 þ 1 min under ultrasonic irradiation), and (ii) coupling reactions, by treatment with a solution of Fmoc-AA(3 eq), HBTU (3 eq), HOBt (3 eq) and DIEA (6 eq) in DMF, and exposing the reagent mixture to the ultrasonic irradiation for 5 min. For the construction of lipidic tails, the amino acid Boc-L-Phe(4-NO 2 )-OH was used and coupled as the last residue to the peptide sequence. Therefore, prior conjugation to lipidic tails, the nitro group was reduced to primary amine by treatment with SnCl 2 1 M solution in DMF, at rt for 16 h. Then, valeric or decanoic acid (3 eq), together with HBTU (3 eq), HOBt (3 eq) and DIEA (6 eq) in DMF/DCM (1:1, v/v), were added to the resin-bound peptides Pent-1B and Dec-1B, respectively, and reactor was agitated on an automated shaker for 2 h. Once the lipidic tail coupling was ascertained by LC-MS analysis, peptides were released from the resin and all protecting groups were simultaneously cleaved by treatment with a cleavage cocktail of TFA/TIS/H 2 O (95:2.5:2.5, v/v/ v), for 3 h under stirring. Finally, lipidated peptides were purified by preparative RP-HPLC equipped with a Phenomenex Kinetex column (C18, 5 mm, 100 Å, 150 Â 4.6 mm) and by using a linear gradient of MeCN (0.1% TFA) in water (0.1% TFA) from 10 to 90% over 30 min, and the correct molecular masses were confirmed through electrospray ionisation mass spectrometry (ESI-MS) analyses (Table S1).

Fluorescein-labelling of peptide dec-1B
The peptide Dec-1B-K(Fam) was labelled to 5(6)-carboxyfluorescein (Fam) on side chain of an additional Lys placed in C-terminus. Thus, Fmoc-L-Lys(Mtt)-OH was introduced during peptide elongation, and, after the construction of the target sequence, as above described, the fluorescein-labelling proceeded as follows. The Mtt group was selectively removed by treating the resin-bound peptide sequence with a cocktail of TFA/TIS/DCM (1:5:94, v/v/v), for 25 min at rt. 16 The procedure was repeated seven times, and the complete deprotection was ascertained by the Kaiser test. The 5(6)-carboxyfluorescein (2 eq) was coupled by using COMU (2 eq), Oxyma (2 eq) and DIPEA (4 eq), and reagent mixture was allowed to stir for 25 min. Then, the resin was washed with DMF (Â 3) and the coupling was repeated for an additional 25 min. The fluorescein labelling was ascertained by the LC-MS analysis. Finally, the peptide was cleaved from the resin and purified by preparative RP-HPLC equipped with a Phenomenex Kinetex column (C18, 5 mm, 100 Å, 150 Â 4.6 mm) and a linear gradient of MeCN (0.1% TFA) in water (0.1% TFA) from 10 to 90% over 30 min, and the correct molecular mass was confirmed by ESI-MS analysis (Table S1).

Determination of the minimum inhibitory concentration (MIC) and the minimum fungicidal concentration (MFC)
The antifungal activity of compounds was determined using a standardised broth microdilution method (Clinical and Laboratory Standards Institute (CLSI) document M27-A4) 34 . Briefly, cell suspension was adjusted to 3 Â 10 3 CFU/mL in RPMI 1640 medium (Sigma) supplemented with 0.2% (w/v) glucose. One hundred microliter aliquots of these cell suspensions were dispensed into 96-well microtiter plates. Compounds were serially diluted using RPMI 1640 medium and added to the wells at a final concentration ranging from 0.4 to 26 lM, and the plate was incubated for 24 h at 37 C. Voriconazole (ranged from 8 to 60 lg/mL for ATCC 10231 and Ca3 strains; ranged from 0.03 lg/mL to 8 lg/mL for other strains) and Amphotericin B (ranged from 0.12 to 2 lg/mL) were chosen as positive controls.
The MIC was defined as the lowest concentration of the peptide that resulted in 100% growth inhibition after 24 h of incubation. The MFC was determined by transferring 50 lL aliquots of  each sample, previously treated with concentrations equal to or higher than the MIC, onto SDA plates and incubating the plates at 37 C for 24 h. The lowest peptide concentration that yielded no fungal growth on agar plates was defined as the MFC. All the tests were conducted at least three times using independent cell suspensions.

Checkerboard assay
The interaction between compounds and voriconazole against voriconazole-resistant C. albicans strains ATCC 10231 and Ca3 clinical strain was evaluated by the checkerboard method in 96-well microtiter plates. Briefly, compounds Pent-1B or Dec-1B and voriconazole were serially diluted along the y and x axes, respectively. The final concentration of voriconazole ranged from 0.03 to 0.5 mg/mL while the final concentration of Pent-1B or Dec-1B ranged from 0.8 to 13 lM for ATCC 10231 and from 0.8 to 26 lM for the clinical strain. The checkerboard plates were inoculated with yeast as previously reported and incubated at 37 C for 24 h, following which yeast growth was assessed visually and the turbidity measured by microplate reader at 595 nm. The Fractional Inhibitory Concentration (FIC) index for each combination was calculated as follows: FIC index ¼ FIC of peptide þ FIC of voriconazole, where FIC of peptide (or voriconazole) was defined as the ratio of MIC of peptide (or voriconazole) in combination and MIC of peptide (or voriconazole) alone. The FIC index values were interpreted as follows: 0.5, synergistic; >0.5 to 1.0, additive; >1.0 to 2.0, indifferent; and >2.0, antagonistic effects 9 .

Peptide cytotoxicity on HaCat cells
To evaluate the effect of Dec-1B on HaCaT cell viability, cells were cultured in Petri culture dishes (100 Â 20 mm) in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS, 2 mM L-glutamine, 100 U mL À1 penicillin, 100 lg mL À1 streptomycin, 25 mM HEPES, (Corning) and 130 lg mL À1 Na pyruvate in a humidified 5% carbon dioxide atmosphere at 37 C. Cell viability was examined as previously described 29 . Briefly, using a colorimetric assay based on the MTT labelling reagent, HaCaT cells (1 Â 10 4 cells/well) were seeded in 96-well plates and, after overnight incubation, were treated with Dec-1B at concentrations of 3.25, 6.50, 13.0, 26.0 mM 35 . As internal control, cells containing only dimethylsulphoxide (DMSO) (Sigma-Aldrich D8418-100 ml) as the solvent at the same concentration used in each test wells were selected. After 24 h, 10 mL of MTT solution (5 mg mL À1 in phosphate-buffered saline, PBS; pH 7.4) was added to each well and the plates were incubated for 3 h at 37 C. Then, the medium was removed, and the obtained formazan crystals were dissolved in 150 lL of DMSO for 15 min. The spectrophotometric absorbance was measured using a microtiter enzyme-linked immunosorbent assay reader (Multiskan TM GO Microplate Spectrophotometer; Thermo Scientific TM ) at 540 nm. The percentage of viability of cells was determined by the following formula: (OD of treated cells/OD of control) Â 100. Our in vitro cytotoxic examination revealed a secure profile for Dec-1B at all tested concentrations on HaCaT cells.

Critical aggregation concentration
The critical aggregation concentration (CAC) of Dec-1B was calculated using Nile Red (NR) as the fluorescent probe. This probe has poor solubility in water, while in presence of hydrophobic environments such as aggregated produces a hyper-hypsochromic effect consisting of a blue-shift and an intensity increase 36 . The peptide stock concentration of 400 mM was prepared dissolving Dec-1B in 1,1,1,3,3,3-Hexafluoro-2-propanol (HFIP), and then different peptide aliquots (0.8, 1, 5, 10, 15, 20, 30, 50, 100, 150, 200, 250, 300 mM) were prepared. The organic solvent was removed under nitrogen stream, 0.5 ml of water was added to each peptide aliquots, and then were sonicated for 15 min and freeze-dried 36 . Then, each peptide solution was hydrated with NR solution of 500 nM for 1 h. The NR spectra were recorded at a fluorescence emission between 570 and 700 nm (slit width, 5 nm), and an excitation wavelength of 550 nm (slit width, 10 nm). All data were analysed by plotting the maximum emission fluorescence corresponding wavelength (y) as a function of the peptide concentration (Â) using the sigmoidal Boltzmann equation as described previously 29 .

Thioflavin T assay
Peptide aggregation in presence of LUVs mimicking fungal membrane was monitored by Thioflavin T (ThT) assay 37 . Once lipid film [PE/PC/PI/Ergosterol (5:4:1:2, w/w/w/w)] at Cf ¼ 100 lM was prepared as described above, it was hydrated with 100 mM NaCl, 10 mM Tris-HCl, 25 mM ThT buffer for 1 h and then was treated through the extrusion method to obtain LUVs. The LUVs were titrated with different peptide concentrations (5, 10, 20, 30 lM) and the ThT emission spectra were recorded before and after the peptide treatment at a fluorescence excitation at 450 nm (slit width, 10 nm) and a fluorescence emission at 482 nm (slit width, 5 nm).

Tryptophan quenching by Br-PC
Three different preparations of large unilamellar vesicles (LUVs) containing 25% of phospholipids 6,7 Br-PC, 9,10 Br-PC, 11,12 Br-PC, were prepared to perform tryptophan quenching experiment being the phospholipid Br-PC known as tryptophan quencher 38 . Lipid films composed of PE/PC/Br-PC/PI/Ergosterol (5:3.7:0.3:1:2, w/ w/w/w/w) were prepared at a final concentration of 125 lM. After the lyophilisation, the film was hydrated with water and LUVs were obtained as described above. The, LUVs were incubated with 5 lM of peptides Pent-1B and Dec-1B for 2 min and the tryptophan spectra emission was recorded setting the fluorescence excitation at 295 nm.

Tryptophan quenching by acrylamide
The tryptophan residue of peptides Pent-1B and Dec-1B was quenched using a 3.25 M solution of acrylamide in the absence and in presence of LUVs composed of PE/PC/PI/Ergosterol (5:4:1:2, w/w/w/w) at a final concentration of 250 lM 39 . Once LUVs were prepared as described above, they were incubated with the peptide at 5 lM for 2 min, and then the tryptophan residue was quenched by acrylamide at different concentrations of 0.02, 0.04, 0.06, 0.08, 0.1, 0.12, 0.14, 0.16, 0.18, and 0.2 M. The same quenching experiment was performed for both peptides in water at a concentration of 5 lM. Fluorescence was measured with k ex 295 nm and k em 340 nm. The data were analysed with the Stern- , where F 0 and F represent the fluorescence intensities in the absence and the presence of the quencher (Q), respectively. The K sv is the Stern-Volmer quenching constant, which is correlated to the accessibility of the tryptophan residue to acrylamide. As acrylamide does not significantly partition into the membrane bilayer, K sv is a reliable reflection of the bimolecular rate constant for collisional quenching of the aromatic residues present in the aqueous phase. K sv is thus determined by the amount of non-lipid-associated free peptide as well as the peptide fraction located on the surface of the bilayer.

NMR spectroscopy
Appropriate amounts of peptides were dissolved in 0.600 ml H 2 O/ D 2 O (90/10 v/v) for a peptide concentration of 0.4-0.8 mM to carry out the NMR analysis in pure water. Then, increasing volumes (lL) of a stock DPC-d 38 were added until no modification was observed on the onedimensional spectra 40 . The NMR analysis in micellar environments was performed at DPC/peptide molar ratio R of 67, corresponding to DPC concentrations of 20-40 mM and peptide concentrations of 0.3-0.6 mM. Assuming a micelle aggregation number of $30, the micelle/peptide molar ratio R is $ 2.
NMR spectra were recorded on a Bruker 700 MHz Spectrometer, located at the Department of Pharmacy -University "Federico II" of Naples, and equipped with a z-gradient 5 mm triple-resonance cryoprobe. One-dimensional proton (1 D) and twodimensional homonuclear (2 D) NMR spectra were recorded at a temperature of 298 K. The spectra were calibrated relative to TSP (0.00 ppm) as an internal standard. 2 D TOCSY (with different mixing times ranging from 25 to 70 ms), and NOESY (mixing times ranging from 100 to 300 ms) spectra were recorded in the phasesensitive mode using the method from States, using 4096 data points in t2 and 512 equidistant t1 values. The water resonance was suppressed by use of gradients. NMR spectra were analysed by using CARA program (http://cara.nmr.ch/doku.php/home). Proton resonances were sequentially assigned by following the Wuthrich standard method 41 and are reported in Tables S2-S5 of the Supplementary Material.

Spin-label experiments
Few microliters of a stock solution of 16-doxylstearic acid (16-DSA) in DMSO-d 6 was added to the DPC peptide samples to obtain a ratio spin-label/micelle 1:1.

Structure determinations
The NOE-based distance restraints were obtained from NOESY spectra recorded with a mixing time of 200 ms. The NOE cross-peaks were integrated with CARA and were converted into upper distance bounds using CALIBA program 42 incorporated into CYANA 43 . Geminal protons were chosen as distance reference with 2.0 Å. Three dimensional structures were obtained by using inter-proton distances evaluated from NOEs as upper limits. Structure calculations started from 100 randomised conformers and used the standard CYANA simulated annealing schedule with 20,000 torsion angle dynamics steps per conformer. As the peptide sequences contain modified residues such as D-residues at positions 9 and 10 (D-Leu 9 and D-Lys 10 ) and the N-terminal phenylalanine (Phe) bearing a long aliphatic chain in position para of phenyl ring i.e. pentanoic acid for Pent-1B and decanoic acid for Dec-1B, all these modifications have been inserted in the special.lib of CYANA suite before calculations. All the conformers showed a fairly agreement with experimental constraints showing no violations.
The best CYANA structures out of 100, in terms of agreement with experimental data, i.e. with lowest target function (TF) values (Table S6), were chosen to represent the peptide molecular structures in solution and visualised by PyMOL software (DeLano (2002) http://www.pymol.org).

Leakage assay
Large unilamellar vesicles (LUVs) mimicking fungal membrane, PE/ PC/PI/Ergosterol (5:4:1:2, w/w/w/w), with ANTs and DPX encapsulated, were prepared as previously described. 21 Firstly, dry lipid film was prepared at a final concentration of 100 mM, and fluorophores ANTs and DPX dissolved in water were added to the film. Then, the lipid preparation was lyophilised overnight. The lipid film loaded with ANTs and DPX was hydrated with PBS 1Â buffer (pH¼ 7.2) for 1 h under stirring and then was treated using the extrusion method to obtain LUVs. In particular, the lipid film with ANTs and DPX was freeze-thawed 6 times and extruded through 0.1-lm pore size polycarbonate filters 10 times. Then, nonencapsulated ANTs and DPX were removed from LUVs through gel filtration using a Sephadex G-50 column (1.5 cm Â 10 cm). In the experiment, we measured the dequenching of ANTs by DPX after the treatment of LUVs with different peptide concentrations of 5, 10, 15, 20, 30, and 50 lM for 15 min. The ANTs fluorescence was recorded at a fluorescence excitation at 385 nm (slit width, 5 nm) and a fluorescence emission at 512 nm (slit width, 5 nm). The complete LUVs leakage and release of ANTs were measured after the treatment of LUVs with a solution of 10% Triton-X for 15 min. Liposomes leakage at different peptide concentrations was calculated as follows: %leakage¼ (F i -F 0 )/(F t -F 0 ), where F 0 is the ANTs fluorescence of the undamaged LUVs, Fi is the ANTs fluorescence after the peptide addition, and F t is the ANTs fluorescence after the Triton-X treatment.

Plasma membrane permeabilization assay
The effect of Pent-1B and Dec-1B on plasma membrane of C. albicans cells was investigated by propidium iodide uptake using LIVE/DEAD FungaLight Yeast Viability Kit (Molecular Probes), according to the methodology by Thevissen et al. with some modifications 43 . A cell suspension of C. albicans ATCC 10231 (200 lL; 3.2 Â 10 6 cells/mL of PBS) was incubated with test peptide at MIC value concentration and live/dead dyes (according to manufacturer instructions) for 30 min and 2 h in microcentrifuges tubes with constant agitation. After this time, the cells were washed, centrifugated (5 min at 10,000 Â g) and suspended in 20 lL of PBS. Images were captured using an LSM 710 inverted confocal laserscanning microscope (Zeiss) equipped with a 63Â objective lens.

Cellular localisation of dec-1B
Confocal laser-scanning fluorescence microscopy was used to study the localisation of Dec-1B in C. albicans ATCC 10231 cells using a double staining 44

Peptide design
Promoting the peptide self-assembly in fungal membrane and reducing the aggregation in aqueous solution through the lipidation strategy could favour an enhancement of the biological activity of AMPs. Different studies have already shown that the addition of fatty acids in N-or C-terminus of AMPs increases their antifungal and antibacterial activities 45,46 . Starting from our previous lipidation study, we identified the para position of Phe 1 of peptide 1B as the best position for the incorporation of lipid tails 29 because when we conjugated fatty acids of variable length in N-terminus or C-terminus of peptide 1B, we observed a loss of activity. In this previous study, we discovered a promising lipidated analogue namely Pent-1B bearing a lipid tail of 5-carbon atoms (C-5) in the para position of Phe 1 (Figure 1), which showed a good biological profile in terms of antimicrobial and cytotoxicity activities.
Herein, we explored the incorporation of a longer lipid tail of 10-carbon atoms (C-10) in the para position of Phe 1 yielding the peptide Dec-1B (Figure 1). The aim of this modification was to enhance the vehiculation and anchoring of the peptide within the fungal membrane, attempting to preserve the good biological profile of peptide Pent-1B 30 . In our design, we kept unchanged the net positive charge of þ4 and the amphipathic properties of the peptide Pent-1B and we did not explore the effect of further longer lipid tails to save the right hydrophobic/hydrophilic balance essential for the membrane interaction and to attain a good biological profile.
Antifungal activity of peptides against C. albicans strains The antimicrobial activity of peptides Pent-1B and Dec-1B was evaluated against C. albicans ATCC 90028 and C. albicans ATCC 10231, and six clinical drug-resistant C. albicans strains. The antimycotic-susceptibility profiles of clinical C. albicans strains are reported in Table 1.
The antifungal activity of peptides, expressed as MIC and MFC values, is shown in Table 2. As reported, both peptides resulted active against both ATCC test strains with MIC values of 13 lM for Pent-1B and 6.5 lM for Dec-1B. The activity tested on clinical strains showed an inhibitory effect on the growth of 4/6 strains for the peptide Pent-1B (MIC values ranged from 26 to 6.5 lM) and of 3/6 strains for the peptide Dec-1B at the highest tested concentration of 26 lM. In addition, the activity of peptides was also investigated in terms of minimum fungicidal concentration.
The MFC values were equal or two-fold higher than the MIC values for Pent-1B, and equal to the corresponding MICs for Dec-1B.

Synergistic effect with voriconazole of acyl-peptides
To further investigate the antifungal activity of Pent-1B and Dec-1B the synergistic interaction with voriconazole on voriconazole-resistant ATCC 10231 and voriconazole-resistant clinical Ca3 strain was determined using the checkerboard technique. After 24 h of incubation we analysed the results to determine the best combination of Pent-1B or Dec-1B with voriconazole. The total absence of growth of the ATCC 10231 strain was observed for     (Table 3).

Peptide effect on HaCaT cell viability
Based on the results from previous experiments, the cytotoxic effect of peptide Dec-1B was tested on human keratinocytes (HaCat cells) using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay in a concentration range from 3.25 to 26 mM. As shown in Figure 2, the peptide Dec-1B did not produce a significant cytotoxic effect on HaCat cells at its antimicrobial concentration of 6.50 mM. Only at the highest concentration of 26 mM, the incorporation of the lipid tails of C-10 caused a slight increase in the cytotoxicity of Dec-1B (75% of cell viability) in comparison with Pent-1B, which showed a negligible cytotoxic effect at highest concentration of 25 mM (86.7% of cell viability in Bellavita et al.) 29 .

Lipid tails promote peptide aggregation in fungal membrane
Lipid tails promote peptide self-assembly in solution as well as in membrane environments. We calculated the critical aggregation concentration (CAC) of peptide Dec-1B in aqueous solution using Nile Red (NR) as fluorescent probe (Figure 3, panel A). NR is not highly soluble in water, and it has a large preference to partition into hydrophobic environments producing a blue shift and hyperchromic effect 36 . Despite the presence of the longer lipid tail (C-10), the peptide Dec-1B has the same CAC of 39.9 mM of the peptide Pent-1B, thus showing similarly a low ability to aggregate in aqueous solution at the concentration used for the antimicrobial assays. We also explored the ability of the peptides to oligomerize in membrane mimetic environments. The peptide Dec-1B, compared to peptide Pent-1B, clearly showed a stronger ability to oligomerize in fungal membranes (Figure 3, panel B). Specifically, we studied peptide aggregation in LUVs composed of PE/PC/PI/Ergosterol (5:4:1:2, w/w/w/w) mimicking fungal membrane and Thioflavin T as fluorescent dye. LUVs were treated with different peptide concentrations (5, 10, 20, and 30 mM). A large enhancement of ThT was recorded at 10 mM for the peptide Dec-1B, revealing a complete peptide aggregation in LUVs. In contrast, for the peptide Pent-1B, the ThT fluorescence at different concentrations indicated a progressive phenomenon of aggregation in LUVs, obtaining an increase of ThT fluorescence at the higher concentration of 30 mM.

Lipopeptides pent-1B and dec-1B are partially inserted into LUVs
The depth of insertion of the peptide into the target membrane is key for activity and for understanding its mechanism of action. We monitored the insertion of lipopeptides Pent-1B and Dec-1B into LUVs mimicking fungal membrane [PE/PC/PI/Ergosterol (5:4:1:2, w/w/w/w)] recording the change of the fluorescence emission of tryptophan (Trp) present in position 4 in both peptides.
We compared the Trp fluorescence emission of peptides in presence of LUVs and in water; furthermore, the accessibility of the Trp residue was measured by quenching it with acrylamide at different concentrations, which is a water-soluble quencher and is unable to insert into the hydrophobic lipid bilayer. As shown in Figure 4, when the peptides Pent-1B and Dec-1B are inserted into hydrophobic environments such as LUVs, the quantum yield of Trp residue changes determining an increase of the Trp fluorescence emission with respect to that recorded in water.
The emission spectra in Figure 4 indicated that both peptides (concentration of 10 mM) were buried in LUVs because the Trp residue resulted to be only partially accessible to the quenching by acrylamide at different concentrations. Being both peptides aggregated at the concentration of 10 mM in LUVs as shown in Figure 3, it is likely that equilibrium phenomena take place between monomeric and oligomeric states which make Trp only partially accessible to quenching by acrylamide. In contrast, when the peptides are in aqueous solution, the Trp is highly accessible because both peptides are in monomeric state at 10 mM as indicated by CAC values (Figure 3).
Stern-Volmer plots for the quenching of tryptophan by acrylamide are shown in Figure 5. In the presence of LUVs, a great decrease in fluorescence intensity was evident, thus revealing that the tryptophan is less accessible to the quencher. In fact, the values for K sv were lower ( Figure 5) in LUVs, confirming that the tryptophan is more buried in the bilayer.
In addition, the localisation of both peptides into LUVs mimicking the fungal membrane was investigated by measuring the quenching of the fluorescence of the Trp residue by the bromo probes bound to the lipid chains of the membrane (Figure 6). We used LUVs composed of 3 different phospholipids bearing bromo quencher in different positions along the hydrocarbon chain. In particular, we used phospholipids 11,12-Br-PC, 9,10-Br-PC, and 6,7-Br-    PC in our experiment. While in 6,7-Br-PC LUVs, the bromo quencher is located at the LUVs interface, in 11,12-Br-PC and 9,10-Br-PC LUVs, the bromo quencher is deeply buried into the lipid core.38 As observed in Figure 5, for both peptides, the largest quenching of Trp fluorescence was recorded in presence of 9,10-Br-PC and 11,12-Br-PC LUVs, while it was less in presence of 6,7-Br-PC LUVs. These results indicated that probably both peptides were inserted into the lipid bilayer and the Trp residue points towards the micellar interior.

NMR conformational analysis shows the localisation of lipopeptides into micelle mimicking membrane environment
We further investigated the interaction and structural properties of peptides Pent-1B and Dec-1B in fungal membranes by NMR spectroscopy using dodecyl-phosphorylcholine (DPC) micelles mimicking membrane environment. NMR studies of Pent-1B and Dec-1B peptides were performed in pure water, where they are perfectly soluble but poorly structured, and in DPC micelles, where they partially adopt helix structures.
In aqueous medium, NMR data, such as the deviations of aCH proton chemical shift ( Figure S1) from random coil values 47 and NOEs collected for Pent-1B and Dec-1B in H 2 O/D 2 O (90/10, v/v) ( Figure S2A and 2C, and Table S6, respectively), indicate random coil conformations of both peptides. This structural diagnosis is confirmed by the molecular models computed by CYANA program 48 using NOE derived distances as upper limit (upl) of interproton distances sketched in Figure S3. The best ten CYANA structures in terms of agreement with experimental data, i.e. with lowest target function (TF) values, were chosen as representative of the conformational space accessible to the peptides and reveal a conformational flexibility of Pent-1B and Dec-1B in pure water, even though some hints of ordered structural elements, bends or turns, emerge in the middle region of peptides. 1 D 1 H spectra of the peptides in water solution as well as the CYANA molecular models obtained for Pent-1B and Dec-1B are shown in Figures  S3, S4A and S4B.
The conformational behaviour of Pent-1B and Dec-1B was also tested in DPC/water mixture (20-40 mM DPC, pH 6, DPC/peptide molar ratio R of 67, DPC micelle/peptide ratio ca. 2). 1 D 1 H spectra of Pent-1B and Dec-1B in DPC are shown in Figure S3A and S5B. In such environment, both peptides adopt a quite ordered structure. The negative aCH deviations from random coil values < À0.1 ppm (Figure S6) point to a helical conformation in the  middle-C-terminal regions of the peptide 46 . This structural diagnosis is consistent with the NOE pattern ( Figure S2B, D).
Indeed, the presence of NOE effects of the type NH i -NH iþ1 , together with long range a i -N iþ2 , a i -N iþ3 and a i -b iþ3 contacts, indicates the occurrence of a helical structure in the central region of the peptides. Structural calculations of Pent-1B and Dec-1B were carried out by CYANA 43 using as upper limit (upl) of inter-proton distances 191 NOE derived distances (127 intra-residues, 33 sequential, 31 long-range) for Pent-1B and 246 NOE derived distances (156 intra-residues, 45 sequential, 45 long-range) for Dec-1B. The statistic of the structural analysis is reported in Table S6. The best CYANA structures were chosen as representative of the conformational space accessible to the peptide (Figure 7).
The backbone superimposition of the best 15 structures shows a well-defined helix arrangement in F 5 -I 12 segment (RMSD value on the backbone atoms of 0.52 Å) that places positively charged and hydrophobic side chains on opposite sides. It is worth noticing that, in this arrangement, all the charged sidechains (K 7 K 10 R 11 ) are iso-oriented as well as the aromatic side chains of N-terminal residues F 1 W 4 and F. 5 Similarly, for Dec-1B the NMR/ CYANA analysis finds a helical arrangement in the segment 5-12 (RMSD value on the backbone atoms of 0.25 Å), with iso-orientation of the hydrophobic and charged side chains (Figure 8).

Spin label studies
The spin label 16-doxyl stearic acid (16-DSA) was added to the peptide/DPC samples to examine the placement of peptides in DPC. 16-DSA is useful for identification of the membraneembedded residues, as it solubilises into DPC micelles 49 . This spin label contains one unpaired electron that induces paramagnetic relaxation resulting in line broadening of resonance signals in proximity to the spin label. A comparison of the cross-peak intensities in the TOCSY spectra of peptide/micelle systems with and without the spin label indicates the residues mostly interacting with micelles.
With a 16-DSA/DPC micelle ratio of 1:1 the cross-peak intensities of the N-terminal residues are strongly reduced, whilst the cross-peak intensities of the C-terminal residues show the same periodic medium or strong reduction for Pent-1B as well as for Dec-1B. The least affected residues are serine 6 and lysine 7 at the beginning of the a-helix structure.
These results suggest that helical domain lies parallel to the micelle surface, with the hydrophobic face towards the interior of the micelle, and the positively charged residues interacting with the negatively charged phosphate headgroups, anchoring the peptide just below the micelle surface ( Figure 9).

Lipopeptides pent-1B and dec-1B did not induce membrane leakage
Since different studies showed that temporins generally act causing the breakdown of bacterial membranes 22,29 , we investigated the mode of action of lipopeptides Pent-1B and evaluated the release of encapsulated ANTs probe in LUVs mimicking fungal membrane [PE/PC/PI/Ergosterol (5:4:1:2, w/w/w/w)]. In these conditions, we did not observe any ANTs release and liposome leakage at different peptide concentrations of 5, 10, 15, 20, 30, and 50 mM (data not shown). Thus, we hypothesised that the mode of action of these lipopeptides may involve an initial electrostatic interaction followed by aggregation and insertion in the lipid bilayer causing perturbation through a carpeting effect as already shown for some temporin analogues 21 and other molecular mechanisms are surely underlying their antifungal activity.

Lipopeptides cause yeast membrane permeabilization
To evaluate the membrane permeabilization of peptides Pent-1B and Dec-1B, C. albicans cells were treated with the LIVE/DEAD FungaLight Yeast Viability kit consisting of labelling dyes such as SYTO 9 and propidium iodide (PI). SYTO 9 penetrates both viable and non-viable cells, while PI only penetrates cells with damaged membranes by turning off the fluorescence emitted by SYTO 9. Damaged or dead cells emit a yellow-red fluorescence, while viable cells emit only green fluorescence. The images were captured by an LSM710 inverted confocal laser scanning microscope (CLSM). All yeast cells showed yellow-red fluorescence after 30 min of treatment with Pent-1B or Dec-1B peptides ( Figure 10, panels B and C), while untreated cells showed green fluorescence ( Figure 10, panel A). The obtained results further support the data obtained by the other experiments and pointing on the ability of both peptides to act on the plasma membrane of these cells, causing a membrane perturbation and thus allowing the entry of the PI dye.

Dec-1B does not enter in yeast mitochondria
Dec-1B labelled with 5(6)-carboxyfluorescein was used to investigate the cellular localisation of the peptide in C. albicans cells using CLSM. A suspension of the ATCC 10231 strain was pre-incubated with MitoTracker Orange and then treated with the labelled peptide at the MIC value concentration. Confocal images showed that the green signal corresponding to the localisation of the fluorescent peptide was uniformly localised on the surface of the treated cells after 30 min. Since the peptide signal has not been shown to overlap the red signal (MitoTracker) of the mitochondria (Figure 11), no localisation in mitochondria seems to occur. The green signal remained localised on yeast surface even after 2 h of treatment. The images therefore confirm that the peptide acts on the fungal wall/cell membranes.

Discussions
The spread of Candida infections and the resistance issues to available antifungal drugs are rapidly increasing. In this scenario, AMPs represent a challenge to open novel avenues and hopes for overcoming the antifungal resistance 14,16 . Previously, using several synthetic strategies on some Temporin L derivatives, including lipidation strategy 29 we developed different analogues which exhibited strong ability to kill both Gram-positive and Gram-negative bacteria, and yeasts as Candida spp 21 . Our previous lipidation study, consisting in the conjugation of fatty acids at the Nand Cterminus of the most promising Temporin L analogue, peptide 1B, allowed to identify the para position of Phe 1 as the best position for performing this functionalization 29 . In this lipidation screening, we discovered the acylated peptide Pent-1B bearing the acyl chain of C-5 on Phe 1 which displayed a strong activity against carbapenem resistant Klebsiella pneumoniae (MIC ¼ 6.25 mM) and a low cytotoxic profile 29,30 . Through a deep study on its potential mode of action, we observed that the presence of the chain of the C-5 in Pent-1B determined a monomeric state in aqueous solution (CAC¼ 39.5 mM) and a significant ability to oligomerize at a low concentration ($10 mM) in LUVs mimicking Gram-positive and Gram-negative membranes 29 . Following the oligomerization in bacterial membrane, Pent-1B induced the pore formation of the target membrane detected by a complete leakage of LUVs mimicking bacterial membrane.
Here, considering these encouraging results, we further investigated the activity of Pent-1B against reference and clinical strains of Candida and we designed and developed its analogue Dec-1B  In general, the interaction of AMPs with the target membranes is regulated by several features, including the length of the sequence, the number and density of positive charges, the secondary structure in aqueous environment and in membranes, and the hydrophobicity 50 . The initial interaction involves the binding to the membrane by a purely non-specific electrostatic interaction between the positively charged residues of the peptide and the negatively charged membrane of bacteria or fungi. Whereas, the peptide hydrophobic domains interact with the hydrophobic core of the lipid bilayer, guiding the peptide deeper into the membrane 50,51 .
In our design, we kept the net positive charge of þ4 of peptide Pent-1B due to Lys and Arg residues and added a longer lipid tail of C-10 to boost the peptide interaction and the internalisation into a complex membrane like that of fungi. The results of antifungal activity on C. albicans ATCC strains (ATCC 10231 and ATCC 90028) showed a significant antifungal activity for Pent-1B and Dec-1B with a MIC of 13 and 6.5 mM, respectively (Table 2). Both peptides were able to reduce the growth of C. albicans clinical strains, with MIC values ranging from 26 lM to 6.5 lM for peptide Pent-1B on Ca1, Ca2, Ca3 and Ca5 strains (Table 2), while the peptide Dec-1B worked at the highest tested concentration of 26 lM on 3 strains (Ca3, Ca4, Ca6). Although in most cases MIC values for clinical  strains were higher than those obtained for ATCC strains, these results seem interesting as the peptides were effective against clinical strains with different resistance profiles, some of which very difficult to treat. In particular, Ca3 and Ca6 strains were resistant to all the azoles, which are widely used for the topical treatment of superficial candidiasis and the systemic therapy of invasive mycoses, while the Ca1 and Ca6 strains were resistant to both azoles and echinocandins, which frequently represent the therapeutic option for azole-resistant Candida strains in hospital settings 52 .
Currently, combination treatment of antifungal drugs with unconventional antifungal agents have been proposed to fight drug-resistant C. albicans infections 53 . In our study, when combined with voriconazole, Dec-1B exhibited a synergistic effect on Ca3 strain, while the association had an additive effect on resistant ATCC strain (Table 3). On the other hand, the combination of Pent-1B with voriconazole was always additive. Noteworthy is the ability of both peptides to significantly reduce the MIC of voriconazole for both resistant strains ATCC 10231 and Ca3, thereby restoring their susceptibility to the antifungal drug. In this case, the combination approach seems particularly advantageous as it allows to recover the effectiveness of drugs such as azoles which have limited toxicity, are cheap and available for oral administration.
Based on these synergistic results, we hypothesised that both peptides may act with different mode of actions compared to conventional antifungal drugs, facilitating the entry of azoles due to their ability to enhance the cell membrane permeability. Clearly, we still cannot exclude that the peptides may also act on some specific molecular pathways and further studies will be necessary to demonstrate this latter mechanism.
We previously showed that Pent-1B acts through a membrane-active mechanism on bacteria 29 thus, here, we explored the mode of action of peptides Pent-1B and Dec-1B through fluorescence assays using liposomes as model systems for mimicking fungal membrane and NMR studies to analyse their three-dimensional structure.
Both peptides were in a monomeric state in aqueous solution at their active antifungal concentrations (around 10 mM) detected by the value of CAC (around 39 mM). Whereas, both peptides showed the ability to oligomerize in liposomes mimicking the Candida membrane detected by the ThT experiment ( Figure 3). In particular, the peptide Dec-1B bearing the longer lipidic tails oligomerized at lower concentration of 10 mM than the peptide Pent-1B that is completely aggregated at 30 mM.
We also analysed through the Br-Trp quenching experiment the depth of internalisation of the peptides in LUVs and the results supported that the tryptophan of both peptides was strongly inserted in the bilayer ( Figure 6). The tryptophan is located at position 4 of the sequence from the N-terminus, which confirms the hypothesis that the flexible domain at the N-terminus is key for the insertion into the bilayer.
Since the other important feature that influences the mode of action of AMPs is three-dimensional structure, we studied the potential mode of action of both peptides by NMR spectroscopy. We observed that both peptides are disordered in water but partially organised in helix when interacting with membrane mimetic systems. The choice of the mimetic system is a critical point in this type of study. We opted for a micellar system instead of bicelles or other heavier mimics because micelles are generally light enough to allow the use of un-labelled (neither 15 N and/or 13 C labelling) cheaper peptides for the NMR interactions studies. The addition of DPC to each Pent-1B and Dec-1B in aqueous solution modifies the NMR spectra. The lines of all signals become larger and some amide protons belonging to the N-terminal segment vanish at all. These effects, by alone, demonstrate that the peptides bind the micelles. Indeed, both peptides change their roto-translation correlation times from those typical of the free forms to those, longer, of the micelle, with consequent higher relaxation rates and larger or vanished signals. Temporin analogue ability to interact with membrane mimics with consequent adoption of helix structure was previously demonstrated 21,22,29 . Here, we showed that the pentanoic or decanoic acid covalently linked to the para position of Phe 1 for Pent-1B and Dec-1B, respectively, favours the N-terminal anchorage of the peptides to the micelle.
For both peptides, the hydrophobic trait, TAIL-F 1 -V 2 -P 3 -W, 4 deeply binds the micelle, while the trait F 5 -L 13 adopts an a-helix structure. The helix arrangement is driven by the contact between the 5-13 trait, amphiphilic in nature, and micelle. The recourse to radical species like 16-doxyl stearic acid (16-DSA), makes it possible to catch the NMR imagine of Pent-1B and Dec-1B docked to the micelle surface. DSA, which solubilises into the micelle, accelerates the nuclear relaxation rates of the nuclei around it. That means that more the peptide side chains are sunk into the micelle, more their signal intensities are diminished. For both Pent-1B and Dec-1B, the nuclei with the most decreased intensities are those of the N-terminal lipophilic segments and those of the first 3-4 residues. The effect of the paramagnetic species on the intensity of the segment 5-13 suggests that the helix lies on the micelle surface, with the aromatic side chains of both Pent-1B ( Figure 9) and Dec-1B iso-oriented towards the micelle interior, while the positively charged sidechains protrude on the opposite side. Thus, the picture emerging by NMR measurements points to peptides that are deeply anchored through the N-terminal segment, while the C-terminal trait lies on the micelle surface fastened by the hydrophobic side chains.
The Br-Trp quenching experiment in membranes mimicking the fungal membrane clearly showed a deeper insertion of the peptides compared to the NMR results obtained into micelles mimicking mammalian membranes, further supporting that the antifungal activity associated by the lack of toxicity to mammalian cells is associated to a different depth of penetration.
The results of the NMR studies in combination with biophysical data clearly showed that both peptides have the typical behaviour of AMPs, characterised by a random conformation in aqueous solution and the adoption of a helical conformation in membrane bilayers. Furthermore, the presence of the lipidic chain favours the peptide anchorage to the membrane and the formation of oligomers inside the membrane. In addition, the deep insertion and the absence of leakage are key to hypothesise a carpet mechanism for the perturbation of the membrane. This assumed mechanism of action of the peptides was further investigated by CLSM. Candida membrane permeabilisation assay showed that 30 min of treatment with Pent-1B or Dec-1B (separately) allowed the uptake of PI suggesting that wall/cell membrane damage and permeabilisation is a possible mode of action of these lipopeptides ( Figure 10). In agreement with these results, the fluorescent labelling of Dec-1B-K(Fam) showed that it remained uniformly localised at the surface of Candida cells and was not localised into mitochondria ( Figure 11).
These data support the hypothesis that Pent-1B and Dec-1B act by interacting and altering the fungal cell membrane as already reported for many antimicrobial peptides, although other mechanisms of action cannot be excluded.

Conclusions
In conclusion, this study elucidated the antifungal activity of our acylated Temporin L analogues Pent-1B and Dec-1B and explored their potential membrane active mechanism. The acylated peptides resulted to be efficacious on both reference and clinical isolates of C. albicans and they showed an additive or synergistic interaction with voriconazole. In addition, both peptides did not show cytotoxicity on human keratinocytes at their antimicrobial concentration. Through a deep study on their potential mechanism of action by using fluorescence-based assays, NMR studies and confocal microscopy, we hypothesised a membrane-active mechanism for peptides Pent-1B and Dec-1B, that consists in an internalisation and oligomerization into fungal membrane, causing a cell membrane damage by a carpeting effect.
These findings may provide insights into the possible therapeutic applications of Temporin analogues as antifungal agents or as enhancers of traditional antifungal drugs that are losing their efficacy due to the development of resistance.